The long term goals of this project are to understand the different stages of T lymphocyte development in terms of signal transduction mediated by the T cell antigen receptor (TCR) and other accessory molecules and co-receptors expressed on the cell surface. TCR ligation in immature thymocytes results in either positive selection, meaning a selection for continued development, or negative selection, the induction of an active cell death program. The proposal is made to investigate the ligands and second messenger signals that result in these two distinct pathways, and compare these cellular responses to those which mediate the immune induction and activation of mature T cells. For these studies TCR transgenic mice will be used. These mice offer a critical advantage in studying cellular signal transduction in different development stages: the majority of thymocytes and T cell express the same TCR. Thus, the induction of cellular responses can be studied using a physiological ligand, antigen peptides bound to class II MHC molecules. Two types of analyses will be carried out. In the first we will compare the signaling responses that can be induced in short-term cultures of thymocytes and T cells. Thymocyte cultures result in the antigen-dependent induction of active cell death, whereas T cell cultures result in blast transformation, IL2 secretion, and proliferation. We will compare the pathways of second messenger signal transduction induced in these cultures, and the requirements for cytokines, and adhesion molecules. In a second set of studies we will study fetal thymic organ cultures (FTOCs) for the conditions that lead to positive vs. negative selection. In these cultures we will examine the effects mediated by different antigenic peptides. This particular TCR transgenic strain is apparently unique in that an MHC allele that can present antigen to these T cells does not, by itself, mediate positive selection in vivo. Thus, in FTOCs we can add different types of peptides at different concentrations, and determine whether we can effect positive or negative selection. In addition we will investigate the effects of various adhesion molecules, and growth factors in TCR-mediated positive and negative selection. Finally, the role of the thymic stroma will be studied. The glycosaminoglycans (GAG) of the cellular and extracellular matrix may play a crucial role in thymic selection and development and this will be studied by manipulating the synthesis and appearance of the GAG matrix in FTOCs.